Introduction of specific mutations into a synthetic internal ribosome entry site (IRESGTX) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P-GTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular enclothelial growth factor 121 (VEGF(121)), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted alpha-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleoticle (resulting in P-GTX2) doubled the SEAP expression levels in CHO-K1. P-GTX2 reached near 70% Of P-SV40-driven expression levels and outperformed constitutive phosphoglycerate kinase (P-PGK) and human ubiquitin C (P-hUBC) promoters in CHO-K1. Also, P-GTX2 was successfully engineered for macrolicle-inducible transgene expression. Owing to its size of only 182 bp, P-GTX2 is one of the smallest eukaryotic promoters. Although P-GTX2 was found to be a potent promoter, it retained its IRESGTX-Specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells. (c) 2006 Wiley Periodicals, Inc.